1. Field of the Invention
This invention relates to a method for the detection of botulinum toxin using bird eggs.
2. Description of the Related Art
Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum block the release of acetylcholine from motor and autonomic nerve termini which leads to flaccid paralysis of afflicted muscles. In mammals paralysis of the diaphragm and intercostal muscles results in ventilatory failure and hypoxia which progresses to brain failure and death. In humans, botulism is primarily caused by ingestion of strains of C. botulinum that produce toxin serotypes A, B, and E and rarely C and F.
The medicinal use of purified type A botulinum toxin (BoNT/A) is expanding at a rapid rate to treat muscle anomalies where hyperactivity results in sustained contractions causing twisted or abnormal posture (strabismus, blepharospasm, torticollis, or achalasia) or where hyperexcitability results in muscle twitching (cervical dystonia, oromandibular dystonia, or spasmodic dystonia) which can be reduced or relieved by muscle paralysis. In addition, BoNT/A is utilized in commercial application for cosmetic uses for the disappearance of frown lines, diminishing facial wrinkles, or intradermal injection to reduce axillary hyperhidrosis. At least ten fold of the medicinal treatment dose would be required to enter the circulatory system to manifest systemic symptoms of botulism in humans (approximately 0.09-0.15 μg intravenously or intramuscularly for a 70-kg human, or a calculated corresponding 70-90 μg oral dose has been extrapolated from trials in primates).
The unintentional ingestion of BoNT in foods has been a health issue for centuries and while botulism in humans is rare it remains a potentially fatal illness. There are typically fewer than 30 cases of foodborne botulism reported annually in the United States. BoNT has also been evaluated as an agent in biological weaponry and is considered the most potent lethal natural toxin known
The initial neurological symptoms of ingested BoNT in humans are ptosis, blurred vision and diplopia followed by dysphonia, dysarthria, and dysphagia. Peripheral muscle weakness consists of descending paralysis affecting first the cranial nerves, then the arms, respiratory muscles, to the legs with onsets from 18 to 36 h after exposure by ingestion. In severe cases, extensive respiratory muscle paralysis leads to ventilatory failure progressing to death unless ventilatory support is provided (<72 h for mice and within 1 to 150 days for humans from the time that symptoms were first detected).
Currently, the mouse toxicity and neutralization bioassay (mouse LD50) is used to analyze samples for the presence of BoNT types A to G activity. In the 16-24 g mouse as little as 10 pg of BoNT/A injected intraperitoneal can be detected within 6 to 96 h. BoNT/A has an LD50 in mice of 1 ng/kg (1 mouse Unit=0.02-0.03 ng BoNT) and is detectable within 72 h. The mouse bioassay is a functionality assay that can detect all 7 biologically active BoNT/A to G. The assay requires a three part approach: toxin screening, toxin titer determination, and finally toxin neutralization using monovalent antibodies for each serotype. This process typically requires 6 days, 2 days of analysis at each part. The potency dosage determination for medicinal BoNT/A with the mouse assay typically utilizes at least 48 mice per test with an additional 48 for the reference sample.(14) There is growing pressure to replace the mouse LD50 assay for ethical concerns over the use of death by asphyxiation of the mice as the test endpoint. There are also scientific concerns over the continued use of a variable animal bioassay for medicinal potency dosage determination when equally as sensitive alternative methods have been reported for simple matrices that may achieve refinement, reduction, and replacement of the use of mice for botulinum toxin texting.
Institutional Animal Care and Use Committee guidelines recommend that animals are to be preemptively euthanized upon observing the earliest onset of severe signs of distress; because severe pain, suffering, or death of experimental animals should be avoided as routine experimental endpoints. LD50 tests are controversial due to the ethics of using a large number of animals and evaluating mainly mortality. Toxicity LD50 testing procedures should incorporate the principles of reduction and refinement until alternative test methods are validated. Preemptive euthanasia of animals should be standard protocol upon exhibiting the progressive signs of a predictable death, an impending death, or when entering into a moribund state. The endpoint for the mouse LD50 assay is commonly death by asphyxiation. In the mouse LD50 assay, when the injected dose is high (as for medicinal potency determination), mice typically develop signs of botulism and death ensues within 8 h. At lower doses, mice are afflicted more slowly; therefore requiring observation every 2 h over the 4 days before negative results are observed and recorded for the assay. The onset of morbidity requires increased frequency of observations, isolation of individual morbid animals, and the removal of dead animals as promptly as possible. The mouse model also inherently has potential safety concerns in that the mice may move during injection leading to the possibility that the researchers could accidentally inject themselves instead of the mouse. Therefore, laboratory personnel who work with BoNTs at unknown dosages on a routine basis and/or inject mice are encouraged to be vaccinated with the botulinum toxoid vaccine. Mice colonies are also expensive to maintain and require daily care and observation. Therefore, complementary models to the mouse would be highly valued.
Currently the mouse assay is used as the model for assessment of botulinum toxin activity. However, the mouse model poses some problems such as the time length needed for the assay (up to 36 hours) as well as safety concerns and requirements for handling needles and live animals which could cause the handler to accidentally self-inject the toxin. While various methods have been developed for the detection of botulinum toxin there remains a need in the art for a quicker method that overcomes the safety issues of the current mouse assay. The present invention's new botulinum toxin detection method of incubating quail embryos overcomes the limitations of the prior art.